Henry L Keith, Field Julie R, Adkins Erika M, Parnas M Laura, Vaughan Roxanne A, Zou Mu-Fa, Newman Amy H, Blakely Randy D
Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
J Biol Chem. 2006 Jan 27;281(4):2012-23. doi: 10.1074/jbc.M505055200. Epub 2005 Nov 3.
In previous studies examining the structural determinants of antidepressant and substrate recognition by serotonin transporters (SERTs), we identified Tyr-95 in transmembrane segment 1 (TM1) of human SERT as a major determinant of binding for several antagonists, including racemic citalopram ((RS)-CIT). Here we described a separate site in hSERT TM3 (Ile-172) that impacts (RS)-CIT recognition when switched to the corresponding Drosophila SERT residue (I172M). The hSERT I172M mutant displays a marked loss of inhibitor potency for multiple inhibitors such as (RS)-CIT, clomipramine, RTI-55, fluoxetine, cocaine, nisoxetine, mazindol, and nomifensine, whereas recognition of substrates, including serotonin and 3,4-methylenedioxymethamphetamine, is unaffected. Selectivity for antagonist interactions is evident with this substitution because the potencies of the antidepressants tianeptine and paroxetine are unchanged. Reduced cocaine analog recognition was verified in photoaffinity labeling studies using [(125)I]MFZ 2-24. In contrast to the I172M substitution, other substitutions at this position significantly affected substrate recognition and/or transport activity. Additionally, the mouse mutation (mSERT I172M) exhibits similar selective changes in inhibitor potency. Unlike hSERT or mSERT, analogous substitutions in mouse dopamine transporter (V152M) or human norepinephrine transporter (V148M) result in transporters that bind substrate but are deficient in the subsequent translocation of the substrate. A double mutant hSERT Y95F/I172M had a synergistic impact on (RS)-CIT recognition ( approximately 10,000-fold decrease in (RS)-CIT potency) in the context of normal serotonin recognition. The less active enantiomer (R)-CIT responded to the I172M substitution like (S)-CIT but was relatively insensitive to the Y95F substitution and did not display a synergistic loss at Y95F/I172M. An hSERT mutant with single cysteine substitutions in TM1 and TM3 resulted in formation of a high affinity cadmium metal coordination site, suggesting proximity of these domains in the tertiary structure of SERT. These studies provided evidence for distinct binding sites coordinating SERT antagonists and revealed a close interaction between TM1 and TM3 differentially targeted by stereoisomers of CIT.
在之前研究血清素转运体（SERTs）对抗抑郁药和底物识别的结构决定因素时，我们确定人SERT跨膜片段1（TM1）中的Tyr-95是几种拮抗剂结合的主要决定因素，包括消旋西酞普兰（(RS)-CIT）。在此我们描述了hSERT TM3中的另一个位点（Ile-172），当它切换为果蝇SERT的相应残基（I172M）时会影响(RS)-CIT的识别。hSERT I172M突变体对多种抑制剂如(RS)-CIT、氯米帕明、RTI-55、氟西汀、可卡因、尼索西汀、马吲哚和诺米芬辛的抑制效力显著丧失，而对包括血清素和3,4-亚甲基二氧甲基苯丙胺在内的底物识别不受影响。由于抗抑郁药噻奈普汀和帕罗西汀的效力未变，因此该取代对拮抗剂相互作用具有选择性。使用[(125)I]MFZ 2-24的光亲和标记研究证实了可卡因类似物识别的降低。与I172M取代不同，该位置的其他取代显著影响底物识别和/或转运活性。此外，小鼠突变体（mSERT I172M）在抑制剂效力方面表现出类似的选择性变化。与hSERT或mSERT不同，小鼠多巴胺转运体（V152M）或人去甲肾上腺素转运体（V148M）中的类似取代导致转运体结合底物，但在随后的底物转运方面存在缺陷。在正常血清素识别的背景下，双突变体hSERT Y95F/I172M对(RS)-CIT识别具有协同作用（(RS)-CIT效力降低约10,000倍）。活性较低的对映体(R)-CIT对I172M取代的反应与(S)-CIT类似，但对Y95F取代相对不敏感，并且在Y95F/I172M处未表现出协同丧失。在TM1和TM3中具有单个半胱氨酸取代的hSERT突变体导致形成高亲和力的镉金属配位位点，表明这些结构域在SERT的三级结构中接近。这些研究为协调SERT拮抗剂的不同结合位点提供了证据，并揭示了CIT立体异构体对TM1和TM3的不同靶向之间的密切相互作用。
